Conventional imaging systems are challenged to provide adequate low-light, high-resolution imaging. Objective components used in high-resolution imaging systems need to have very high numeric aperture (NA) values. Unfortunately, a high NA value of the objective component results in a very small depth of field in which to view target objects. A small depth of field raises significant challenges in achieving and maintaining focus of target objects to be viewed during low-light, high-resolution imaging. If focus of a target object is not achieved and maintained, the resultant defocused image of the target object at a detector is spread over an unacceptably large area of the detector, with a loss in spatial resolution and a decrease in the signal-to-noise ratio associated with the image of the target object.
Confocal microscopy provides the ability to image cross sections of a cell (“optical sectioning”) for the purpose of generating a three-dimensional map of cellular structures, or to synthesize a single two-dimensional image in which all cellular structures are in focus. These capabilities are desirable for a wide range of cell analysis applications, including co-localization studies, quantifying the translocation of molecules between cellular compartments, and the enumeration of fluorescence in situ hybridization probes randomly located in a nucleus. Although confocal microscopy provides a highly detailed view of the cell, the repeated scanning required significantly reduces image acquisition rates, and can in some cases, induce photo-bleaching of fluorescent probes.
Currently confocal microscopy is limited by the length of time required to capture imagery, the types of signals that can be collected simultaneously, and the limitation that the cells be immobilized on a solid support. The relatively slow speed of confocal microscopy can be a limiting factor for many applications. Commonly-studied cellular phenomena, including signaling, internalization of surface-bound factors, chromosomal defects, and various morphological transformations, can be subject to high cell-to-cell variation, occur over a wide and continuous range of values, or occur at low frequencies within a heterogeneous mixture of cells. Therefore, the study of such phenomena can require the observation and analysis of thousands of cells, and the application of statistical analysis in order to reach robust and repeatable scientific conclusions. In such cases, it is often impractical to employ confocal microscopy, due to the low throughput of the technique, despite the wealth of information it can provide for each cell.
In the alternative, conventional fluorescence imaging is generally much faster than confocal image stacking and can provide good spatial resolution and fluorescence sensitivity, when employing high NA objectives. However, conventional fluorescence microscopy is subject to a tradeoff between NA and depth of field. As the NA is increased to improve light collection and increase spatial resolution, the depth of field is reduced by the square of the NA change. Therefore, images of weakly fluorescent signals and cellular structures located outside the ideal plane of focus can be compromised. This effect is most readily observed in experiments employing Fluorescence In Situ Hybridization (FISH) probes that are typically under one micron in size and are comprised of a limited number of fluorescent molecules, which can be distributed throughout the nucleus or cytoplasm of a cell. A slight defocus may preclude the detection of dim probes, or cause multiple probes located in close proximity to blur into each other. Larger amounts of defocus can cause substantial blur, rendering a FISH spot unrecognizable in an image. These tradeoffs for increased speed over the highly focused imagery produced by confocal image stacking are generally not acceptable, given that conventional microscopy, even in automated form, is still slow compared to flow cytometry. As a result, many studies of cellular phenomena employ both flow cytometry (for the high throughput study of large cell populations) and confocal microscopy (for the detailed imaging of selected individual cells).
The ImageStream™ flow imaging system was developed in part to address the gap between the slow, but detailed information obtained by confocal microscopy and the fast, but limited cellular information gleaned by flow cytometry. The ImageStream™ system collects six simultaneous multi-mode images (brightfield, darkfield, and up to four different fluorescence colors) from cells in flow. High fluorescence sensitivity and resolution are achieved by using 0.75 NA optics and a 0.5 micron pixel size.
Several attempts have been made to extend the depth of field of such a flow imaging system. For example, U.S. Pat. No. 6,583,865 (the disclosure and drawings of which are hereby specifically incorporated herein by reference) describes the use of a flow imaging system having a tilted detector (or a sample flow path that is tilted relative to the detector) that effectively increases the depth of field for a more accurate enumeration of structures and probes within a cell. The technique can be used in connection with a pulsed light source to produce multiple images of a moving object at different focal planes, or it can employ a continuous light source to produce a single composite image incorporating information from the object at multiple focal planes. The pulsed light source variant is limited in fluorescence sensitivity because each image has a relatively short signal integration time. The continuous light source variant is limited in image quality because the composite image contains both in-focus and out-of-focus information at every location in the cell. Hence, there is a need for a high speed imaging system having an extended depth of field as well as both high fluorescence sensitivity and excellent image quality.
U.S. Pat. No. 7,009,651 (the disclosure and drawings of which are hereby also specifically incorporated herein by reference) describes a flow imaging system in which light from an object is split into a plurality of optical paths, and one or more of the optical paths are defocused relative to the default focal plane of the system, to similarly increase the depth of field. U.S. Pat. No. 6,211,955 (the disclosure and drawings of which are hereby also specifically incorporated herein by reference) describes the use of a stereoscopic imaging apparatus to view cells from multiple angles, for the reconstruction of a three-dimensional (3-D) map of the cell and accurate enumeration of FISH spots in images. The effectiveness of this technique is limited by the depth of field that can be achieved with the imaging system. If the depth of field of each detector is less than the depth of the cell, or at least, of the nucleus, the spatial resolution of the three-dimensional map produced by the technique will vary across the cell, and neighboring FISH spots in the image will blur into each other and be unresolved.
While the ImageStream™ flow imaging system represents a significant advance over conventional flow cytometry and standard microscopy, demanding applications, such as the quantization of FISH probed cells, require imaging capabilities closer to those achieved by confocal image stacking.
It would therefore be desirable to develop a flow imaging system suitable for high-resolution imagery (0.75 NA and 0.5 micron pixel size), which also exhibits an extended depth of field.